S-phenenyltris (iminocarbonyl) triisophthalic acid salts

ABSTRACT

s-Phenenyltris(iminocarbonyl)triisophthalic acid salts useful as complement inhibitors.

DESCRIPTION OF THE INVENTION

This invention is concerned with compounds of the formula: ##STR1## wherein R is selected from the group consisting of alkali metal; and the pharmaceutically acceptable salts thereof.

The compounds of the present invention may be prepared by treating a mixture of 3,5-carbomethoxy benzoyl chloride and N,N-dimethylaniline in acetonitrile with 1,3,5-triaminobenzene for 16 hours. The product is isolated as the hexamethyl ester. This ester may be converted to the hexasodium salt by treatment with sodium hydroxide.

BACKGROUND OF THE INVENTION

The term "complement" refers to a complex group of proteins in body fluids that, working together with antibodies or other factors, play an important role as mediators of immune allergic, immunochemical and/or immunopathological reactions. The reactions in which complement participates take place in blood serum or in other body fluids, and hence are considered to be humoral reactions.

With regard to human blood, there are at present more than 11 proteins in the complement system. These complement proteins are designated by the letter C and by number: C1, C2, C3 and so on up to C9. The complement protein C1 is actually an assembly of subunits designated C1q, C1r and C1s. The numbers assigned to the complement proteins reflect the sequence in which they become active, with the exception of complement protein C4, which reacts after C1 and before C2. The numerical assignments for the proteins in the complement system were made before the reaction sequence was fully understood. A more detailed discussion of the complement system and its role in body processes can be found in, for example, Bull. World Health Org., 39, 935-938 (1968); Ann. Rev. Medicine, 19, 1-24 (1968); The John Hopkins Med. J., 128, 57-74 (1971); Harvey Lectures, 66, 75-104 (1972); The New England Journal of Medicine, 287, 452-454; 489-495; 545-549; 592-596; 642-646 (1972); Scientific American, 229, (No. 5), 54-66 (1973); Federation Proceedings, 32, 134-137 (1973); Medical World News, October 11, 1974, pp. 53-58; 64-66; J. Allergy Clin. Immunol., 53, 298-302 (1974); Cold Spring Harbor Conf. Cell Proliferation 2/Proteases Biol. Control/229-241 (1975); Annals of Internal Medicine, 84, 580-593 (1976); "Complement: Mechanisms and Functions", Prentice-Hall, Englewood Cliffs, N.J. (1976).

The complement system can be considered to consist of three sub-systems: (1) a recognition unit (C1q) which enables it to combine with antibody molecules that have detected a foreign invader; (2) an activation unit (C1r, C1s, C2, C4, C3) which prepares a site on the neighboring membrane; and (3) and attack unit (C5, C6, C7, C8 and C9) which creates a "hole" in the membrane. The membrane attack unit is non-specific; it destroys invaders only because it is generated in their neighborhood. In order to minimize damage to the host's own cells, its activity must be limited in time. This limitation is accomplished partly by the spontaneous decay of activated complement and partly by interference by inhibitors and destructive enzymes. The control of complement, however, is not perfect, and there are times when damage is done to the host's cells. Immunity is therefore a double-edged sword.

Activation of the complement system also accelerates blood clotting. This action comes about by way of the complement-mediated release of a clotting factor from platelets. The biologically active complement fragments and complexes can become involved in reactions that damage the host's cells, and these pathogenic reactions can result in the development of immune-complex diseases. For example, in some forms of nephritis, complement damages the basal membrane of the kidney, resulting in the escape of protein from the blood into the urine. The disease disseminated lupus erythematosus belongs in this category; its symptoms include nephritis, visceral lesions and skin eruptions. The treatment of diphtheria or tetanus with the injection of large amounts of antitoxin sometimes results in serum sickness, an immune-complex disease. Rheumatoid arthritis also involves immune complexes. Like disseminated lupus erythematosus, it is an autoimmune disease in which the disease symptoms are caused by pathological effects of the immune system in the host's tissues. In summary, the complement system has been shown to be involved with inflammation, coagulation, fibrinolysis, antibody-antigen reactions and other metabolic processes.

In the presence of antibody-antigen complexes the complement proteins are involved in a series of reactions which may lead to irreversible membrane damage if they occur in the vicinity of biological membranes. Thus, while complement constitutes a part of the body's defense mechanism against infection it also results in inflammation and tissue damage in the immunopathological process. The nature of certain of the complement proteins, suggestions regarding the mode of complement binding to biological membranes and the manner in which complement effects membrane damage are discussed in Annual Review in Biochemistry, 38, 389 (1969).

A variety of substances have been disclosed as inhibiting the complement system, i.e., as complement inhibitors. For example, the compounds 3,3'-ureylenebis-[6-(2-amino-8-hydroxy-6-sulfo-1-naphthylazo)]benzenesulfonic acid, tetrasodium salt (chlorazol fast pink), heparin and a sulphated dextran have been reported to have an anticomplementary effect, British Journal of Experimental Pathology, 33, 327-339 (1952). The compound 8-(3-benzamido-4-methylbenzamido)naphthalene-1,-3,5-trisulfonic acid (Suramin) is described as a competitive inhibitor of the complement system, Clin. Exp. Immunol., 10, 127-138 (1972). German Pat. No. 2,254,893 or South African Pat. No. 727,923 discloses certain 1-(diphenylmethyl)-4-(3-phenylallyl)piperazines useful as complement inhibitors. Other chemical compounds having complement inhibiting activity are disclosed in, for example, Journal of Medicinal Chemistry, 12, 415-419; 902-905; 1049-1052; 1053-1056 (1969); Canadian Journal of Biochemistry, 47, 547-552 (1969); The Journal of Immunology, 93, 629-640 (1964); The Journal of Immunology, 104, 279-288 (1970); The Journal of Immunology, 106, 241-245 (1971) and The Journal of Immunology, 111, 1061-1066 (1973).

It has been reported that the known complement inhibitors epsilon-aminocaproic acid, Suramin and tranexamic acid all have been used with success in the treatment of hereditary angioneurotic edema, a disease state resulting from an inherited deficiency or lack of function of the serum inhibitor of the activated first component of complement (C1 inhibitor), The New England Journal of Medicine, 286, 808-812 (1972). It has also been reported that the drug, pentosan-poly-sulfoester, has an anticomplementary activity on human serum both in vitro and in vivo, as judged by the reduction in total hemolytic complement activity; Pathologie Biologie, 25, 33-36 (1977).

SPECIFIC DISCLOSURE EXAMPLE 1 5,5',5"-[s-Phenenyltris(iminocarbonyl)]triisophthalic acid hexamethyl ester

To a stirred mixture of 6.25 g of 3,5-carbomethoxy benzoyl chloride and 2.98 g of N,N-dimethylaniline in 50 ml of acetonitrile is added 1.0 g of 1,3,5-triaminobenzene in 30 ml of acetonitrile with formation of a solid. An additional 180 ml of acetonitrile is added and stirring is continued overnight. The solid is collected, washed with acetonitrile and ether and air dried. The material is slurried in hot ether, filtered and dried, then is slurried in hot water, filtered, washed with ethanol and ether and oven dried to yield 5.6 g of the product of the Example as an off-white solid.

EXAMPLE 2 5,5',5"-[s-Phenenyltris(iminocarbonyl)]triisophthalic acid hexasodium salt

A 391 mg portion of the product of Example 1 is added to 10 ml of dimethylformamide and 0.4 ml of 10N sodium hydroxide. Solution is achieved by warming and stirring, some water is added and stirring is continued at room temperature for one/half hour. The precipitate formed is filtered, washed with ethanol and ether and dried. This material is slurried in ethanol, filtered, washed and dried, then is reprecipitated from 10 ml of water with ethanol to give the product of the Example which is collected, washed and dried as above to yield 260 mg of a light tan solid.

EXAMPLE 3 Preparation of Compressed Tablet

    ______________________________________                                         Ingredient              mg/Tablet                                              ______________________________________                                         Active Compound         0.5-500                                                Dibasic Calcium Phosphate N.F.                                                                         qs                                                     Starch USP              40                                                     Modified Starch         10                                                     Magnesium Stearate USP  1-5                                                    ______________________________________                                    

EXAMPLE 4 Preparation of Compressed Tablet - Sustained Action

    ______________________________________                                         Ingredient            mg/Tablet                                                ______________________________________                                         Active Compound       0.5-500 (as acid                                         as Aluminum Lake*, Micronized                                                                        equivalent)                                              Dibasic Calcium Phosphate N.F.                                                                       qs                                                       Alginic Acid          20                                                       Starch USP            35                                                       Magnesium Stearate USP                                                                               1-10                                                     ______________________________________                                          *Complement inhibitor plus aluminum sulfate yields aluminum complement         inhibitor. Complement inhibitor content in aluminum lake ranges from           5-30%.                                                                   

EXAMPLE 5 Preparation of Hard Shell Capsule

    ______________________________________                                         Ingredient           mg/Capsule                                                ______________________________________                                         Active Compound      0.5-500                                                   Lactose, Spray Dried qs                                                        Magnesium Stearate   1-10                                                      ______________________________________                                    

EXAMPLE 6 Preparation of Oral Liquid (Syrup)

    ______________________________________                                         Ingredient             % W/V                                                   ______________________________________                                         Active Compound        0.05-5                                                  Liquid Sugar           75.0                                                    Methyl Paraben USP     0.18                                                    Propyl Paraben USP     0.02                                                    Flavoring Agent        qs                                                      Purified Water qs ad   100.0                                                   ______________________________________                                    

EXAMPLE 7 Preparation of Oral Liquid (Elixir)

    ______________________________________                                         Ingredient             % W/V                                                   ______________________________________                                         Active Compound        0.05-5                                                  Alcohol USP            12.5                                                    Glycerin USP           45.0                                                    Syrup USP              20.0                                                    Flavoring Agent        qs                                                      Purified Water qs ad   100.0                                                   ______________________________________                                    

EXAMPLE 8 Preparation of Oral Suspension (Syrup)

    ______________________________________                                         Ingredient            % W/V                                                    ______________________________________                                         Active Compound       0.05-5                                                   as Aluminum Lake, Micronized                                                                         (acid equivalent)                                        Polysorbate 80 USP    0.1                                                      Magnesium Aluminum Silicate,                                                   Colloidal             0.3                                                      Flavoring Agent       qs                                                       Methyl Paraben USP    0.18                                                     Propyl Paraben USP    0.02                                                     Liquid Sugar          75.0                                                     Purified Water qs ad  100.0                                                    ______________________________________                                    

EXAMPLE 9 Preparation of Injectable Solution

    ______________________________________                                         Ingredient            % W/V                                                    ______________________________________                                         Active Compound       0.05-5                                                   Benzyl Alcohol N.F.   0.9                                                      Water for Injection   100.0                                                    ______________________________________                                    

EXAMPLE 10 Preparation of Injectable Oil

    ______________________________________                                         Ingredient           % W/V                                                     ______________________________________                                         Active Compound      0.05-5                                                    Benzyl Alcohol       1.5                                                       Sesame Oil qs ad     100.0                                                     ______________________________________                                    

EXAMPLE 11 Preparation of Intra-Articular Product

    ______________________________________                                         Ingredient              Amount                                                 ______________________________________                                         Active Compound         2-20 mg                                                NaCl (physiological saline)                                                                            0.9%                                                   Benzyl Alcohol          0.9%                                                   Sodium Carboxymethylcellulose                                                                          1-5%                                                   pH adjusted to 5.0-7.5                                                         Water for Injection qs ad                                                                              100%                                                   ______________________________________                                    

EXAMPLE 12 Preparation of Injectable Depo Suspension

    ______________________________________                                         Ingredient            % W/V                                                    ______________________________________                                         Active Compound       0.05-5                                                                         (acid equivalent)                                        Polysorbate 80 USP    0.2                                                      Polyethylene Glycol 4000 USP                                                                         3.0                                                      Sodium Chloride USP   0.8                                                      Benzyl Alcohol N.F.   0.9                                                      HCl to pH 6-8         qs                                                       Water for Injection qs ad                                                                            100.0                                                    ______________________________________                                    

The compounds of the present invention may be administered internally, e.g., orally, intra-articularly or parenterally, e.g., intra-articular, to a warm-blooded animal to inhibit complement in the body fluid of the animal, such inhibition being useful in the amelioration or prevention of those reactions dependent upon the function of complement, such as inflammatory process and cell membrane damage induced by antigen-antibody complexes. A range of doses may be employed depending on the mode of administration, the condition being treated and the particular compound being used. For example, for intravenous or subcutaneous use from about 5 to about 50 mg/kg/day, or every six hours for more rapidly excreted salts, may be used. For intra-articular use for large joints such as the knee, from about 2 to about 20 mg/joint per week may be used, with proportionally smaller doses for smaller joints. The dosage range is to be adjusted to provide optimun therapeutic response in the warm-blooded animal being treated. In general, the amount of compound administered can vary over a wide range to provide from about 5 mg/kg to about 100 mg/kg of body weight of animal per day. The usual daily dosage for a 70 kg subject may vary from about 350 mg to about 3.5 g. Unit doses of the acid or salt can contain from about 0.5 mg to about 500 mg.

While in general the sodium salts of the acids of the invention are suitable for parenteral use, other salts may also be prepared, such as those of primary amines, e.g., ethylamine; secondary amines, e.g., diethylamine or diethanol amine; tertiary amines, e.g., pyridine or triethylamine or 2-dimethylaminomethyl-dibenzofuran; aliphatic diamines, e.g., decamethylenediamine; and aromatic diamines, can be prepared. Some of these are soluble in water, others are soluble in saline solution, and still others are insoluble and can be used for purposes of preparing suspensions for injection. Furthermore as well as the sodium salt, those of the alkali metals, such as potassium and lithium; of ammonia; and of the alkaline earth metals, such as calcium or magnesium, may be employed. It will be apparent, therefore, that these salts embrace, in general derivatives of salt-forming cations.

In therapeutic use, the compounds of this invention may be administered in the form of conventional pharmaceutical compositions. Such compositions may be formulated so as to be suitable for oral or parenteral administration. The active ingredient may be combined in admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, i.e., oral or parenteral. The compounds can be used in compositions such as tablets. Here, the principal active ingredient is mixed with conventional tabletting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums, or similar materials as non-toxic pharmaceutically acceptable diluents or carriers. The tablets or pills of the novel compositions can be laminated or otherwise compounded to provide a dosage form affording the advantage of prolonged or delayed action or predetermined successive action of the enclosed medication. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids or mixtures of polymeric acids with such materials as shellac. shellac and cetyl alcohol, cellulose acetate and the like. A particularly advantageous enteric coating comprises a styrene maleic acid copolymer together with known materials contributing to the enteric properties of the coating. The tablet or pill may be colored through the use of an appropriate non-toxic dye, so as to provide a pleasing appearance.

The liquid forms in which the novel compositions of the present invention may be incorporated for administration include suitable flavored emulsions with edible oils, such as, cottonseed oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar pharmaceutical vehicles. Sterile suspensions or solutions can be prepared for parenteral use. Isotonic preparations containing suitable preservatives are also desirable for injection use.

The term dosage form, as described herein, refers to physically discrete units suitable as unitary dosage for warm-blooded animal subjects, each unit containing a predetermined quantity of active component calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specification for the novel dosage forms of this invention are indicated by characteristics of the active component and the particular therapeutic effect to be achieved or the limitations inherent in the art of compounding such an active component for therapeutic use in warm-blooded animals as disclosed in this specification. Examples of suitable oral dosage forms in accord with this invention are tablets, capsules, pills, powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing and other forms as herein described.

The complement inhibiting activity of the compounds of this invention has been demonstrated by one or more of the following identified tests: (i) Test, Code 026 (C1 inhibitor)-This test measures the ability of activated human C1 to destroy fluid phase human C2 in the presence of C4 and appropriate dilutions of the test compound. An active inhibitor protects C2 from C1 and C4; (ii) Test, Code 035 (C3-C9 inhibitor)-This test determines the ability of the late components of human complement (C3-C9) to lyse EAC 142 in the presence of appropriate dilutions of the test compound. An active inhibitor protects EAC 142 from lysis by human C3-C9; (iii) Test, Code 036 (C-Shunt inhibitor) - In this test human erythrocytes rendered fragile are lysed in autologous serum via the shunt pathway activated by cobra venom factor in the presence of appropriate dilutions of the test compound. Inhibition of the shunt pathway results in failure of lysis; (iv) Forssman Vasculitis Test -- Here, the well known complement dependent lesion, Forssman vasculitis, is produced in guinea pigs by intradermal injection of rabbit anti-Forssman antiserum. The lesion is measured in terms of diameter, edema and hemorrhage and the extent to which a combined index of these is inhibited by prior intraperitoneal injection of the test compound at 200 mg/kg is then reported, unless otherwise stated; (v) Forssman Shock Test -- Lethal shock is produced in guinea pigs by an i.v. injection of anti-Forssman antiserum and the harmonic mean death time of treated guinea pigs is compared with that of simultaneous controls; (vi) Complement Level Reduction Test -- In this test, the above dosed guinea pigs, or others, are bled for serum and the complement level is determined in undiluted serum by the capillary tube method of U.S. Pat. No. 3,876,376 and compared to undosed control guinea pigs; and (vii) Cap 50 Test -- Here, appropriate amounts of the test compound are added to a pool of guinea pig serum in vitro, after which the undiluted serum capillary tube assay referred to above is run. The concentration of compound inhibiting 50% is reported.

With reference to Table I, guinea pigs weighing about 300 g were dosed intravenously (i.v.) or intraperitoneally (i.p.) with 200 mg/kg of the test compound dissolved in saline and adjusted to pH 7-8. One hour after dosing, the guinea pigs were decapitated, blood was collected and the serum separated. The serum was tested for whole complement using the capillary tube assay. Percent inhibition was calculated by comparison with simultaneous controls. The results appear in Table I together with results of tests, code 026, 035, 036, Cap 50, % inhibition and Forssman shock. Table I shows that the compounds of the invention possess highly significant in vitro and in vivo, complement inhibiting activity in warm-blooded animals.

Some of the compounds of the present invention have been found to possess anti-coagulant activity as well as complement inhibiting activity. The in vitro anti-coagulant activity (AC) of the compounds of this invention has been demonstrated by the following test: Citrated sheep plasma (CSP) is added to various dilutions of test compound in a Microtiter® plate, the CSP sample mixtures are then recalcified with an isotonic sheep red blood cell (RBC) suspension. The sheep RBC's, kept in suspension throughout the clotting incubation time, become enmeshed in the fibrin matrix if a clot forms. Upon centrifugation of the plate, untrapped RBC's form buttons, the sizes of which correspond to the degree of clot inhibition; this providing a measure of anti-coagulant activity (AC). Sodium heparin is used as a positive control and activity is reported in wells appearing in Table I.

                                      TABLE I                                      __________________________________________________________________________     Biological Activities                                                                         Cl  C-Late                                                                             Shunt-Inhibition                                                       026*                                                                               035*                                                                               036*     Cap                                                                               AC*                                         Compound       Wells                                                                              Wells                                                                              Wells    50*                                                                               Wells                                       __________________________________________________________________________     5,5',5"-[s-Phenenyltris(imino-                                                 carbonyl)]triisophthalic acid                                                                 +3**                                                                               N   N        328                                                                               <-2                                         hexasodium salt                                                                __________________________________________________________________________      *Code designation for tests employed as referred herein.                       **Activity in wells a serial dilution assay. Higher well number indicates      higher activity. The serial dilutions are two-fold.                      

The computation of an Intrinsic Therapeutic Index (ITI) was devised to correlate the results expressed in wells, obtained in the in vitro Code 026 (C1 inhibitor) test and the in vitro anti-coagulant (AC) test into a meaningful value which would aid in the net evaluation of the activity of the compounds of this invention.

The ITI of a given compound may be defined as the antilogarithm of the logarithmic (base 2) difference between the highest serial dilution in wells which is active in the Code 026 test and the highest serial dilution in wells providing activity in the anti-coagulant test. The ITI is thus a measure of the separation of anti-complement and anti-coagulant activities; the higher the numerical value the more therapeutically useful the separation of activities.

The Intrinsic Therapeutic Index of the compounds of this invention are listed in Table II.

                                      TABLE II                                     __________________________________________________________________________     Intrinsic Therapeutic Index                                                                   In Vitro Activity                                                                                   Logarithmic                                               Complement Inhibiting                                                                      Anti-Coagulant                                                                          Difference                                                                            Intrinsic                                          Activity (Wells)                                                                           Activity (Wells)                                                                        Expressed as                                                                          Therapeutic                         Compound       Code 026    AC       Wells  Index                               __________________________________________________________________________     5,5',5"-[s-Phenenyltris-                                                       (iminocarbonyl)]triisophthalic                                                                +3          -2       +5     32                                  acid hexasodium salt                                                           __________________________________________________________________________ 

We claim:
 1. A compound of the formula: ##STR2## wherein R is selected from the group consisting of alkali metal; and the pharmaceutically acceptable salts thereof. 